A simple and non-invasive method for analyzing local immune responses in vivo using fish fin

Accepted Manuscript A simple and non-invasive method for analyzing local immune responses in vivo using fish fin Yuta Matsuura, Naoki Takaoka, Ryuichiro Miyazawa, Teruyuki Nakanishi, PhD PII:

S0145-305X(17)30162-3

DOI:

10.1016/j.dci.2017.04.016

Reference:

DCI 2880

To appear in:

Developmental and Comparative Immunology

Received Date: 26 March 2017 Revised Date:

19 April 2017

Accepted Date: 19 April 2017

Please cite this article as: Matsuura, Y., Takaoka, N., Miyazawa, R., Nakanishi, T., A simple and non-invasive method for analyzing local immune responses in vivo using fish fin, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.04.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Title: A simple and non-invasive method for analyzing local immune responses in vivo using fish fin

RI PT

Author names:

Yuta Matsuuraa, Naoki Takaokaa, Ryuichiro Miyazawaa, Teruyuki Nakanishia, *

a

SC

Affiliations:

Department of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa 252-0880,

M AN U

Japan

*Corresponding author; Teruyuki Nakanishi, PhD

Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University,

TE D

1866 Kameino, Fujisawa, Kanagawa, 252-0880, Japan Tel.: +81-466-84-3383; fax: +81-466-84-3380

AC C

EP

E-mail address: [email protected] (T. Nakanishi)

1

ACCEPTED MANUSCRIPT

Immunocompetence is an important parameter that reflects disease resistance in fish.

2

Very few methods to examine immunocompetence in vivo have been developed, even

3

for mammals. In the present study, we present a unique method for analyzing local

4

immune responses using fish fin. We first demonstrated the migration of granulocytes to

5

the site of zymosan injection in fin in a dose-dependent manner and that this could be

6

easily observed macroscopically due to the fin membrane transparency. We also

7

demonstrated phagocytic activity of accumulated leukocytes after zymosan

8

administration and that almost all phagocytes were granulocytes. In addition, we

9

succeeded to detect respiratory burst activity of granulocytes in vivo without any in vitro

M AN U

SC

RI PT

1

treatment of cells, indicating that our present method is suitable for the analysis of

11

granulocyte phagocytic function in vivo. The method provides a unique tool applicable

12

for fishes that possess transparent fins and may lead to better understanding of the

13

mechanisms of local immune responses in fish.

Key words: teleost; fin; NBT assay; respiratory burst activity; local immunity

EP

15

AC C

14

TE D

10

ACCEPTED MANUSCRIPT

1 2

1. Introduction Immunocompetence, defined as the capacity to mount an effective immune response following an infection, is a critical parameter for disease resistance. To analyze

4

immunocompetence in vivo, several methods have been established. Tuberculin skin

5

testing (also known as Mantoux testing) has been used for the diagnosis of tuberculosis

6

infection in humans (Vukmanovic-Stejic et al., 2006). The test endpoint involves a

7

classical delayed-type hypersensitivity (DTH) response that reflects the systemic

8

cell-mediated immune response. A phytohaemagglutinin (PHA) skin test has been

9

developed to evaluate cell-mediated immunity in birds (Tella et al., 2008). The swelling

M AN U

SC

RI PT

3

caused by PHA injection reflects the systemic T cell-mediated immune response.

11

Salaberria et al., (2013) proposed that this test is also applicable to investigate the

12

functional activity of phagocytes. Furthermore, a skin window technique has been used

13

to investigate the acute inflammatory response in vivo in humans and monkeys

14

(Maguire et al., 2010; Rebuck and Crowley, 1955). This technique involves creation of

15

a lesion in the stratum corneum of the skin and enables visualization of leukocyte

16

migration.

EP

17

TE D

10

As in mammals, phagocytosis in fish is accomplished by professional phagocytes, e.g. macrophages and neutrophils, although phagocytic activity of B cells in fish has

19

also been reported (Oriol Sunyer, 2012). The phagocytic process includes a series of

20

interactive sequential events including migration, phagocytosis and respiratory burst

21

activity. Most in vitro experiments have been conducted focusing independently on each

22

biological event, ignoring the interactions between them. To address this problem, in

23

vivo studies on host-parasite interactions employing live imaging techniques have

24

recently been developed using transparent zebrafish embryos and larvae (Meeker and

AC C

18

1

ACCEPTED MANUSCRIPT

25

Trede, 2008; Trede et al., 2004). However, the live imaging technique is not applicable

26

to adult zebrafish due to the lack of transparency. Thus a new model is required for in

27

vivo investigation of the mechanisms of phagocytosis in adult fish. In the present study, we developed a novel method to analyze responses of immune

RI PT

28

cells in vivo using transparent fish fin. We first examined the migration/accumulation of

30

leukocytes to the site of antigen/stimulant injection. Administration of zymosan into fin

31

membrane resulted in migration of granulocytes to the injection site in a dose-dependent

32

manner. We demonstrated that the migration and/or accumulation of the granulocytes

33

could be observed macroscopically due to the transparency of fin membrane. We then

34

investigated the phagocytic activity of granulocytes in vivo following injection of

35

zymosan. Finally, we succeeded to demonstrate the respiratory burst activity of

36

granulocytes in vivo without any in vitro treatment of cells, indicating that our present

37

method is suitable for the analysis of granulocyte phagocytic function in vivo. The

38

method provides a unique tool applicable to fish that possess a transparent fin and may

39

lead to better understanding of the mechanisms of local immune responses in fish.

40

EP

TE D

M AN U

SC

29

2. Materials and Methods

42

2.1. Fish

43

AC C

41

The fish used in this study were an isogeneic clone of ginbuna crucian carp from the

44

island of Okushiri (OB1 clone). Fish, weighing 20-25 g, were maintained in 800 L tanks

45

with running water at 26±1 °C and fed twice daily with commercial pellets.

46 47 48

2.2. Monoclonal antibody Flow cytometry analysis utilized a culture supernatant of a hybridoma that produces 2

ACCEPTED MANUSCRIPT

49

the mouse monoclonal antibody (mAb) GB21 that recognizes

50

granulocytes/macrophages (Shibasaki et al., 2015). The immunostaining patterns of the

51

antibody are shown in Supplemental Fig. 2, 3B and 3C.

53 54

RI PT

52

2.3. Administration of reagents into fin

Fish were anesthetized with 50 mg/L benzocaine (Sigma-Aldrich, St Louis, MO) and reagents were injected into compartments which consisted of membranes of the

56

dorsal fin using insulin syringes with 29G needles (Terumo, Tokyo, Japan). In this study,

57

we termed the method as ‘‘intra-fin administration’’ (illustrated in Supplemental Fig. 1).

58

The following reagents were injected into the base of four continuous compartments of

59

dorsal fin in a total volume of 100 µL: 1:10 dilution of fluorescent beads (1.0 µm

60

Fluoresbrite Carboxylate Microspheres, Polysciences, Inc., Warrington, PA), each

61

concentration of zymosan A (Sigma-Aldrich), 1 mg/mL pHrodo®-labeled Green

62

Zymosan Bioparticles (Thermo Fisher Scientific Inc., Waltham, MA) and 0.2%

63

Nitroblue Tetrazolium (NBT, Wako Pure Chemical, Osaka, Japan). pHrodo® is a

64

pH-sensitive dye that increases fluorescence intensity in response to decreasing pH

65

during phagocytosis (Foukas et al., 1998). NBT is a tetrazolium salt that is converted to

66

a deep purple, water-insoluble formazan product upon reduction by superoxide (Pick,

67

1986). All reagents were dissolved in phosphate buffered saline (PBS).

69

M AN U

TE D

EP

AC C

68

SC

55

2.4. Observation of fin

70

Fish fin was visualized using a digital camera for macroscopic analysis and an

71

Olympus SZX12 stereomicroscope with Olympus DP73 digital camera and software

72

(Olympus, Tokyo, Japan) for microscopic analysis, respectively. 3

ACCEPTED MANUSCRIPT

73 74

2.5. Preparation of fin leukocytes Fish were deeply anesthetized with 50 mg/L benzocaine and their spinal cords were

76

severed for euthanasia. Fin tissue including the administration site was cut from the fin

77

base and disaggregated by pressing through a 100-gauge mesh stainless steel sieve in

78

Hank’s Balanced Salt Solution (Nissui Pharmaceutical co., Tokyo, Japan) supplemented

79

with 0.5% heat-inactivated FBS (0.5% FBS-HBSS).

2.6. Cytospin preparation

SC

81

M AN U

80

RI PT

75

Two hundred microliters of 5 x 105 cells /mL of fin cell suspension was centrifuged

83

at 100 × g for 5 min at room temperature onto a glass slide using cytospin and air-dried.

84

For bright field illumination, prepared specimens were stained with May-Grunwald

85

Giemsa. For fluorescence observation, the specimens were fixed with 4%

86

paraformaldehyde (Wako Chemicals, Osaka, Japan) for 15 min at room temperature.

87

The specimens were then washed three times using PBS and nuclei were stained with 1

88

µg/mL DAPI (Thermo Fisher Scientific Inc.). The specimens were then mounted with

89

ProLong Gold anti-fade mounting medium (Thermo Fisher Scientific Inc.).

91 92

EP

AC C

90

TE D

82

2.7. Analysis of fin granulocytes For immunostaining, cells prepared as described in section 2.5 were washed twice in

93

0.5% FBS-HBSS by centrifugation at 500 × g for 5 min at 4 °C and incubated with

94

mAb GB21 diluted 1:10 for 1 hour at 4 °C. A monoclonal mouse IgG2a antibody

95

(eBioscience, San Diego, CA) was used as a primary isotype-matched control. The cells

96

were then washed three times with the medium and incubated with a 1:500 dilution of 4

ACCEPTED MANUSCRIPT

Alexa 647 donkey anti-mouse IgG antibody (Thermo Fisher Scientific Inc.) for 30 min

98

at 4°C. The cells were then washed twice and suspended in 0.2 mL of the medium

99

containing 2.5 µg/mL propidium iodide (Thermo Fisher Scientific Inc.). The mAb

100

GB21+ granulocytes were identified by FACS Canto gated on the FSCmed SSChigh

101

population excluding dead cells (Becton Dickinson and Company, Franklin Lakes, NJ).

103 104

2.8. Microscopic observation of fin granulocytes

SC

102

RI PT

97

Specimens stained with May-Grunwald Giemsa were observed using an Olympus BX-51 microscope with an Olympus DP20 digital camera and software (Olympus).

106

For fluorescence observation, fluorescence microscopy (Olympus IX71) was used with

107

a digital camera and software (Olympus DP73).

M AN U

105

108

111

TE D

110

2.9. Statistics

All statistical analyses were performed using Graph Pad Prism 6 version 6 (Graph Pad Software, San Diego, CA).

112

EP

109

3. Results

114

3.1. Visualization of fluorescent beads injected into fin membrane

115

AC C

113

We first demonstrated that fish fin is suitable as an administration site, and injected

116

fluorescence beads were visible through the transparent fin membrane for at least 3 days

117

following administration (Fig. 1A, B, C).

118

3.2. Migration of granulocytes to fin tissue following zymosan administration

119 120

Intra-fin administration of zymosan resulted in migration of granulocytes to the injection site in a dose-dependent manner (Fig. 2A, B, C). The cells reached a peak at 5

ACCEPTED MANUSCRIPT

6-12 hours after administration and decreased at 24 hours (Fig. 2D). Interestingly,

122

swelling of fin membrane that accompanied granulocyte migration was easily observed

123

after administration (Fig. 3).

124

3.3. Kinetics of phagocytosing granulocytes in fin tissue following zymosan

125

administration

RI PT

121

Microscopic observation of fin cells isolated from zymosan-administrated fish

127

showed that cells that accumulated at the fin phagocytosed zymosan particles (Fig. 4A,

128

B). In addition, analysis using pHrodo-labeled particles revealed that almost all of the

129

phagocytic cells were GB21+ granulocytes (Fig. 5A, B). Phagocytosing granulocytes

130

were present at 6 hours and the percentage of phagocytosing cells among granulocytes

131

significantly increased at 12 hours after administration, and remained at high levels

132

through 48 hours (Fig. 5C). These results confirmed that our method could be used for

133

in vivo analysis of phagocytosis by granulocytes.

134

3.4. Reduction of NBT in the fin following zymosan administration

M AN U

TE D

135

SC

126

Administration of NBT and zymosan together into fin membrane changed the color of the zymosan to deep purple, and the change was clearly visible to the naked eye

137

through the transparent fin membrane (Fig. 6C, D). In addition, microscopic analysis of

138

isolated cells from the fin showed that granulocytes contained deep purple precipitates

139

derived from reduced formazan in the zymosan particles (Fig. 7A, B) indicating the

140

occurrence of respiratory burst activity in vivo in the fin.

AC C

EP

136

141 142 143 144

4. Discussion In the present study, we present a new method for analyzing local immune responses in vivo using the fish dorsal fin. We demonstrated that the migrated and/or accumulated 6

ACCEPTED MANUSCRIPT

granulocytes to the site of zymosan injection could be observed macroscopically due to

146

the transparency of fin membrane. We also succeeded to demonstrate the phagocytic

147

activity and the respiratory burst activity of granulocytes in vivo following injection of

148

zymosan. These results indicate that our present method is suitable for the analysis of

149

granulocyte phagocytic function in vivo.

RI PT

145

The intra-fin administration method is non-invasive and can be utilized for in situ

151

observation analogous to the ‘‘dorsal skin window chamber system’’ established in mice

152

(Palmer et al., 2011). That system has been used to study angiogenesis, tumor

153

microenvironments and treatment responses and allows optical access to broad areas

154

without surgical treatment. However, the chamber system requires special surgical

155

techniques to create the chamber, and surgical stress is a concern. Furthermore, in our

156

system it was easy to remove the tissue and isolate the cells from the fish fin. Thus, our

157

intra-fin administration method is superior to the chamber system.

M AN U

TE D

158

SC

150

Granulocytes are key players in the inflammatory response against a variety of pathogens. These cells are thought to play important roles in protection during the early

160

phases of infection in fish (Ellis, 1999; Secombes and Fletcher, 1992). Havixbeck et al.,

161

(2017) reported that an increase in neutrophils at the wound surface contributed to

162

killing and pathogen elimination during early stages of infection of goldfish challenged

163

with Aeromonas veronii. We analyzed granulocyte kinetics using our present method,

164

with zymosan as a model inflammatory substance (Doherty et al., 1985; Gado and

165

Gigler, 1991; Xie et al., 2010). We first demonstrated that injected fluorescence beads

166

were visible through the transparent fin membrane for at least 3 days following injection.

167

We also demonstrated the dose-dependent migration of granulocytes to the injection site.

168

Therefore, our method is suitable for analyzing the migration of granulocytes and

AC C

EP

159

7

ACCEPTED MANUSCRIPT

169 170

applicable for optical assessment of granulocyte migration toward stimulants in vivo. Once phagocytosed, foreign particles such as pathogens are destroyed by production of reactive oxygen species (ROS) in the cytoplasm of phagocytes (Laskay et al., 2003).

172

NADPH oxidase, a major ROS generator, induces a burst of oxygen consumption called

173

the ‘‘respiratory burst’’(Segal and Abo, 1993) that is essential for killing a number of

174

microorganisms (Hampton et al., 1998). Although the analysis of granulocyte function

175

in vitro has been performed with cells obtained from fish peritoneal cavity (Havixbeck

176

et al., 2016; Rieger et al., 2012; Serada et al., 2005) or swim bladder (Matsuyama and

177

Iida, 1999), results obtained in vitro do not always reflect the true function in vivo.

178

Accordingly, our study is the first in vivo observation of the biological function of the

179

cells, in contrast to in vitro assays. Furthermore, in the present study we could observe

180

the occurrence of respiratory burst in phagocytosing granulocytes through the

181

transparent fin membrane by the change of color of zymosan particles when NBT was

182

added. We are now attempting to establish a quantitative method to analyze the activity

183

in situ using a colorimetric assay.

SC

M AN U

TE D

Increase in phagocytic activity has been used as an indicator of immunocompetence

EP

184

RI PT

171

with respect to host innate immunity (Anderson, 1992; Sakai et al., 1989; Secombes,

186

1994; Yin et al., 2009). With our new methods it is simple and easy to analyze the

187

phagocytic activity of cells in vivo. Furthermore, the methods have the advantage of

188

allowing analysis of local as opposed to systemic immune responses. In tuberculin skin

189

testing and PHA skin tests, the only information obtained is the degree of tissue

190

swelling. Although cellular analysis of immune cells is possible using the window

191

chamber technique, it requires surgical stress and associated trauma. In contrast, in fish

192

fin tissue the accumulated cells are visible with the naked eye and easily collected from

AC C

185

8

ACCEPTED MANUSCRIPT

193

the site of stimulant injection. Taken together, our method is superior to previous

194

techniques widely used in mammals.

195

The present results showed that migration of granulocytes to the fin occurred soon after zymosan injection and reached a peak at 6 - 12 hours. In other studies, infiltration

197

of granulocytes peaked at 18 - 24 hours after in vivo injection into the peritoneal cavity

198

(Chadzinska et al., 2009; Havixbeck et al., 2016; Rieger et al., 2012) and swim bladder

199

(Matsuyama and Iida, 1999). These results indicate that there is a distinct difference in

200

immune cell recruitment between the fin, which is directly exposed to the environment,

201

and internal organs. Because the fin is an important portal of entry for viruses (Costes et

202

al., 2009; Harmache et al., 2006) and bacteria (Khimmakthong et al., 2013;

203

Marco-Noales et al., 2001), it has been considered to be the first line of defense against

204

such pathogens. Accordingly, previous reports on infiltration of granulocytes into

205

peritoneal cavity and swim bladder may not reflect true immune mechanisms at the

206

portal of entry for microorganisms. Furthermore, we demonstrated that phagocytosis did

207

not occur immediately after migration, because a peak of phagocytosis occurred at least

208

six hours after the peak of migration. Several in vitro analyses of phagocytosis report

209

that it occurred rapidly and reached a peak within 1 hour after starting the assay

210

(Esteban et al., 1998; Rodriguez et al., 2003). In mammals, interleukin-8 (IL-8), which

211

is produced in inflammatory sites, is essential for neutrophil activation (Harada et al.,

212

1994). However, the involvement of cytokines has not been taken into consideration in

213

the in vitro experiments. Our method enabled us to demonstrate the consecutive

214

wholistic biological events of immune cells at a portal of pathogen entry.

215 216

AC C

EP

TE D

M AN U

SC

RI PT

196

Our present method should prove useful for many fish species, including important aquaculture species, that possess transparent fin. No special tools are needed. We are 9

ACCEPTED MANUSCRIPT

currently investigating adaptation of the present method to the analysis of other types of

218

immune cells, such as lymphocytes. In addition, the present method may be useful for

219

analysis of the interaction between pathogens and host leukocytes, employing live

220

imaging techniques. Our method allows for the analysis in adult fish that possess

221

sufficient numbers of leukocytes to examine gene expression analysis, functional

222

analysis, etc. Furthermore, gut and gill are gaining attention recently as sites for

223

mucosal immunity in fish. Fish fin may be an additional site for the study of local and

224

mucosal immune responses.

SC

In conclusion, we developed a simple and non-invasive method for analyzing local

M AN U

225

RI PT

217

immune responses in vivo using fish fin. In the present study we demonstrated the

227

migration and the function of granulocytes in fin tissue in vivo. Our present method is

228

applicable to other fish species and other cell types as mentioned above. Importantly,

229

our method allows us to visulalize the migration of cells and their function through

230

transparent fin membrane. Furthermore, the present method may be useful for analysis

231

of the interaction between pathogens and host leukocytes employing live imaging

232

techniques.

EP

233

TE D

226

Acknowledgements

235

This study was supported in part by a Sasakawa Scientific Research Grant from The

236

Japan Science Society and a Grant-in-Aid for Scientific Research (B) (Grant Number

237

16H04984) from the Japan Society for the Promotion of Science (JSPS).

AC C

234

238 239

References

240

Anderson, D.P., 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: 10

ACCEPTED MANUSCRIPT

Applications to aquaculture. Annu. Rev. Fish Dis. 2, 281-307.

242

Chadzinska, M., Savelkoul, H.F., Verburg-van Kemenade, B.M., 2009. Morphine affects

243

the inflammatory response in carp by impairment of leukocyte migration. Dev. Comp.

244

Immunol. 33, 88-96.

245

Costes, B., Raj, V.S., Michel, B., Fournier, G., Thirion, M., Gillet, L., Mast, J., Lieffrig,

246

F., Bremont, M., Vanderplasschen, A., 2009. The major portal of entry of koi

247

herpesvirus in Cyprinus carpio is the skin. J. Virol. 83, 2819-2830.

248

Doherty, N.S., Poubelle, P., Borgeat, P., Beaver, T.H., Westrich, G.L., Schrader, N.L.,

249

1985. Intraperitoneal injection of zymosan in mice induces pain, inflammation and the

250

synthesis of peptidoleukotrienes and prostaglandin E2. Prostaglandins 30, 769-789.

251

Ellis, A.E., 1999. Immunity to bacteria in fish. Fish Shellfish Immunol. 9, 291-308.

252

Esteban, M.A., Mulero, V., Munoz, J., Meseguer, J., 1998. Methodological aspects of

253

assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream

254

(Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue. Res. 293,

255

133-141.

256

Foukas, L.C., Katsoulas, H.L., Paraskevopoulou, N., Metheniti, A., Lambropoulou, M.,

257

Marmaras, V.J., 1998. Phagocytosis of Escherichia coli by insect hemocytes requires

258

both activation of the Ras/mitogen-activated protein kinase signal transduction pathway

259

for attachment and beta3 integrin for internalization. J. Biol. Chem. 273, 14813-14818.

260

Gado, K., Gigler, G., 1991. Zymosan inflammation: a new method suitable for

261

evaluating new antiinflammatory drugs. Agents and actions 32, 119-121.

262

Hampton, M.B., Kettle, A.J., Winterbourn, C.C., 1998. Inside the neutrophil

263

phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007-3017.

264

Harada, A., Sekido, N., Akahoshi, T., Wada, T., Mukaida, N., Matsushima, K., 1994.

AC C

EP

TE D

M AN U

SC

RI PT

241

11

ACCEPTED MANUSCRIPT

Essential involvement of interleukin-8 (IL-8) in acute inflammation. J. Leukoc. Biol. 56,

266

559-564.

267

Harmache, A., LeBerre, M., Droineau, S., Giovannini, M., Bremont, M., 2006.

268

Bioluminescence imaging of live infected salmonids reveals that the fin bases are the

269

major portal of entry for Novirhabdovirus. J. Virol. 80, 3655-3659.

270

Havixbeck, J.J., Rieger, A.M., Churchill, L.J., Barreda, D.R., 2017. Neutrophils exert

271

protection in early Aeromonas veronii infections through the clearance of both bacteria

272

and dying macrophages. Fish Shellfish Immunol. 63, 18-30.

273

Havixbeck, J.J., Rieger, A.M., Wong, M.E., Hodgkinson, J.W., Barreda, D.R., 2016.

274

Neutrophil contributions to the induction and regulation of the acute inflammatory

275

response in teleost fish. J. Leukoc. Biol. 99, 241-252.

276

Khimmakthong, U., Deshmukh, S., Chettri, J.K., Bojesen, A.M., Kania, P.W., Dalsgaard,

277

I., Buchmann, K., 2013. Tissue specific uptake of inactivated and live Yersinia ruckeri

278

in rainbow trout (Oncorhynchus mykiss): visualization by immunohistochemistry and in

279

situ hybridization. Microb. Pathog. 59-60, 33-41.

280

Laskay, T., van Zandbergen, G., Solbach, W., 2003. Neutrophil granulocytes--Trojan

281

horses for Leishmania major and other intracellular microbes? Trends Microbiol. 11,

282

210-214.

283

Maguire, S., Dobrzynski, E., Alpaugh, R.K., 2010. Modified skin-window technique in

284

cynomolgus macaques (Macaca fasicularis) for assessing neutrophil extravasation. J.

285

Am. Assoc. Lab. Anim. Sci. 49, 475-479.

286

Marco-Noales, E., Milan, M., Fouz, B., Sanjuan, E., Amaro, C., 2001. Transmission to

287

eels, portals of entry, and putative reservoirs of Vibrio vulnificus serovar E (biotype 2).

288

Appl. Environ. Microbiol. 67, 4717-4725.

AC C

EP

TE D

M AN U

SC

RI PT

265

12

ACCEPTED MANUSCRIPT

Matsuura, Y., Takasaki, M., Miyazawa, R., Nakanishi, T., 2017. Stimulatory effects of

290

heat-killed Enterococcus faecalis on cell-mediated immunity in fish. Dev. Comp.

291

Immunol. 74, 1-9.

292

Matsuyama, T., Iida, T., 1999. Degranulation of eosinophilic granular cells with possible

293

involvement in neutrophil migration to site of inflammation in tilapia. Dev. Comp.

294

Immunol. 23, 451-457.

295

Meeker, N.D., Trede, N.S., 2008. Immunology and zebrafish: spawning new models of

296

human disease. Dev. Comp. Immunol. 32, 745-757.

297

Oriol Sunyer, J., 2012. Evolutionary and functional relationships of B Cells from fish

298

and mammals: Insights into their novel roles in phagocytosis and presentation of

299

particulate antigen. Infect. Disord. Drug Targets 12, 200-212.

300

Palmer, G.M., Fontanella, A.N., Shan, S.Q., Hanna, G., Zhang, G.Q., Fraser, C.L.,

301

Dewhirst, M.W., 2011. In vivo optical molecular imaging and analysis in mice using

302

dorsal window chamber models applied to hypoxia, vasculature and fluorescent

303

reporters. Nat. Protoc. 6, 1355-1366.

304

Pick, E., 1986. Microassays for superoxide and hydrogen peroxide production and

305

nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader.

306

Methods Enzymol. 132, 407-421.

307

Rebuck, J.W., Crowley, J.H., 1955. A method of studying leukocytic functions in vivo.

308

Ann. NY Acad. Sci. 59, 757-805.

309

Rieger, A.M., Konowalchuk, J.D., Grayfer, L., Katzenback, B.A., Havixbeck, J.J.,

310

Kiemele, M.D., Belosevic, M., Barreda, D.R., 2012. Fish and mammalian phagocytes

311

differentially regulate pro-inflammatory and homeostatic responses in vivo. PloS one 7,

312

e47070.

AC C

EP

TE D

M AN U

SC

RI PT

289

13

ACCEPTED MANUSCRIPT

Rodriguez, A., Esteban, M.A., Meseguer, J., 2003. Phagocytosis and peroxidase release

314

by seabream (Sparus aurata L.) leucocytes in response to yeast cells. Anat. Rec. A

315

Discov. Mol. Cell Evol. Biol. 272, 415-423.

316

Sakai, M., Atsuta, S., Kobayashi, M., 1989. Protective Immune Response in Rainbow

317

Trout, Oncorhynchus mykiss, Vaccinated with β-haemolytic Streptococcal

318

Bacterin. Fish Pathol. 24, 169-173.

319

Salaberria, C., Muriel, J., de Luna, M., Gil, D., Puerta, M., 2013. The PHA test as an

320

indicator of phagocytic activity in a passerine bird. PloS one 8, e84108.

321

Secombes, C.J., 1994. Enhancement of fish phagocyte activity. Fish Shellfish Immunol.

322

4, 421-436.

323

Secombes, C.J., Fletcher, T.C., 1992. The role of phagocytes in the protective

324

mechanisms of fish. Annu. Rev. Fish Dis. 2, 53-71.

325

Segal, A.W., Abo, A., 1993. The biochemical basis of the NADPH oxidase of

326

phagocytes. Trends Biochem. Sci. 18, 43-47.

327

Serada, K., Moritomo, T., Teshirogi, K., Itou, T., Shibashi, T., Inoue, Y., Nakanishi, T.,

328

2005. Comparison of respiratory burst activity of inflammatory neutrophils in ayu

329

(Plecoglossus altivelis) and carp (Cyprinus carpio). Fish Shellfish Immunol. 19,

330

363-373.

331

Shibasaki, Y., Matsuura, Y., Toda, H., Imabayashi, N., Nishino, T., Uzumaki, K.,

332

Hatanaka, C., Yabu, T., Moritomo, T., Nakanishi, T., 2015. Kinetics of lymphocyte

333

subpopulations in allogeneic grafted scales of ginbuna crucian carp. Dev. Comp.

334

Immunol. 52, 75-80.

335

Tella, J.L., Lemus, J.A., Carrete, M., Blanco, G., 2008. The PHA test reflects acquired

336

T-cell mediated immunocompetence in birds. PloS one 3, e3295.

AC C

EP

TE D

M AN U

SC

RI PT

313

14

ACCEPTED MANUSCRIPT

Trede, N.S., Langenau, D.M., Traver, D., Look, A.T., Zon, L.I., 2004. The use of

338

zebrafish to understand immunity. Immunity 20, 367-379.

339

Vukmanovic-Stejic, M., Reed, J.R., Lacy, K.E., Rustin, M.H., Akbar, A.N., 2006.

340

Mantoux Test as a model for a secondary immune response in humans. Immunol. Lett.

341

107, 93-101.

342

Xie, K., Yu, Y., Zhang, Z., Liu, W., Pei, Y., Xiong, L., Hou, L., Wang, G., 2010.

343

Hydrogen gas improves survival rate and organ damage in zymosan-induced

344

generalized inflammation model. Shock 34, 495-501.

345

Yin, G., Ardo, L., Thompson, K.D., Adams, A., Jeney, Z., Jeney, G., 2009. Chinese

346

herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp,

347

Cyprinus carpio, and protection against Aeromonas hydrophila. Fish. Shellfish.

348

Immunol. 26, 140-145.

M AN U

SC

RI PT

337

TE D

349

Figure legends

351

Fig. 1. Visualization of fluorescent beads injected into fin membrane.

352

Macroscopic observation of fin tissue was performed under visible light (A) and

353

ultra-violet light (B). Microscopic observation was performed using stereoscopy.

354

Fluorescence image (C) is shown. All observations were performed three days after

355

administration of fluorescent beads.

AC C

356

EP

350

357

Fig. 2. Migration of granulocytes to fin tissue following zymosan administration.

358

(A, B) Flow-cytometry analysis of fin cells. PBS (A) and 500 µg zymosan (B) was

359

administered. Circles indicate granulocytes populations (FSCmed SSChigh).

360

(C) Dose-dependent changes (percentages) in accumulated granulocytes following 15

ACCEPTED MANUSCRIPT

administration of different amounts of zymosan. The ratio of granulocytes (GB21+ cells

362

gated on FSCmed SSChigh) to other cells isolated from fin is presented. Statistical

363

significance was calculated using one-way ANOVA, followed by Dunnett's multiple

364

comparisons test. (**, p<0.01). n=4. Mean ±S.E.M.

365

(D) Time course analysis of granulocyte population after administration of 100 µg

366

zymosan (closed circles) or PBS (open circles). Statistical significance was calculated

367

using two-way ANOVA, followed by Sidak's multiple comparisons test. (****,

368

p<0.0001). n=4. Mean ±S.E.M.

SC

M AN U

369

RI PT

361

Fig. 3. Accumulation of leukocytes at the site of zymosan injection.

371

PBS (left panel) or 500 µg zymosan (right panel) was administered into fin, and

372

macroscopic observation of fin tissue was performed under visible light at each time

373

point after administration. Arrow indicates the injection site.

TE D

370

374

Fig. 4. Microscopic analysis of phagocytosing cells isolated from fin tissue.

376

Cytospin preparations of cells isolated from the fin of a zymosan (100 µg) administered

377

fish are shown. Bar represents 50 µm (A) and 10 µm (B) in the image (original

378

magnification x 10 (A) and x 40 (B)).

AC C

379

EP

375

380

Fig. 5. Kinetics of phagocytosing granulocytes in fin tissue following zymosan

381

administration.

382

(A, B) Flow-cytometry analysis of phagocytosing granulocytes using pHrodo labeled-

383

zymosan. PBS (A) or 100 µg of pHrodo labeled-zymosan (B) was injected into fin and

384

the accumulated cells were harvested 24 hours after administration. 16

ACCEPTED MANUSCRIPT

(C) Time course analysis of phagocytosing granulocytes in fin tissue following zymosan

386

administration. Cells were isolated from fin tissue at each time point following

387

administration of 100 µg pHrodo-labeled zymosan (closed circles) or PBS (open circles).

388

The ratio of pHrodo-labeled zymosan+ cells to all GB21+ granulocytes is shown.

389

Statistical significance was calculated using two-way ANOVA, followed by Sidak's

390

multiple comparisons test. (**, p<0.01; ***, p<0.001; ****, p<0.0001). n≥3. Mean

391

±S.E.M.

SC

RI PT

385

392

Fig. 6. Reduction of NBT in the fin following zymosan administration.

394

Fin was observed under visible light at 24 hours after administration of 0.2% NBT with

395

(B) or without (A) 100 µg zymosan.

396

M AN U

393

Fig. 7. Microscopic analysis of phagocytic granulocytes containing reduced NBT.

398

GB21+ fin cells isolated 24 hours after administration of 0.2% NBT (A) or 0.2% NBT

399

with 100 µg zymosan (B, C) were observed by using fluorescence microscopy (original

400

magnification x 40 in A and B, x 60 in C). An isotype-matched control prepared using

401

fin cells isolated 24 hours after administration of 0.2% NBT with 100 µg zymosan is

402

shown (D). Nuclei were stained with DAPI (blue) and GB21+ cells were visualized by

403

Alexa 594 (red). Arrow and arrowhead indicate a zymosan particle stained by reduced

404

NBT and a non-stained zymosan particle, respectively. Bars represent 10 µm in each

405

image.

AC C

EP

TE D

397

406 407

Supplemental Fig. 1 Illustration of the ‘‘intra-fin administration’’ method.

408

Dorsal fin and the site of injection (*) are shown. Twenty five microliters of reagents 17

ACCEPTED MANUSCRIPT

409

were administrated at each injection site (*) for a total of 100 µl.

410

Supplemental Fig. 2

412

(A) FSC/SSC dot plots of fin leukocytes. Fin cells were isolated from fish administered

413

500 µg of zymosan. Granulocytes and myeloid cells were gated on FSCmed SSChigh and

414

FSChigh SSClow populations, respectively.

415

(B, C) Immunostaining pattern of mAb GB21-labeled cells in granulocyte (B) and

416

myeloid (C) fractions.

SC

RI PT

411

M AN U

417

Supplemental Fig. 3

419

(A) FSC/SSC dot plots of kidney leukocytes. Granulocytes, lymphocytes and myeloid

420

cells were gated on FSCmed SSChigh, FSClow SSClow and FSChigh SSClow populations,

421

respectively.

422

(B, C) Morphology of GB21+ cells.

423

GB21+ cells gated on FSCmed SSChigh (B) and FSChigh SSClow (C) population were sorted

424

and stained with May-Grunwald Giemsa (MG) and Sudan Black B (SBB). Bars represent

425

10 µm in each image. Isolation of kidney leukocytes and subsequent cell sorting was

426

performed using naive fish according to the methods of Matsuura et al. (2017).

AC C

EP

TE D

418

18

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 2

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 3

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 4

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 5

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 6

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 7

ACCEPTED MANUSCRIPT

Highlights


A simple and unique method for analyzing local immune responses using fish fin in vivo was

RI PT

developed.


Migration of granulocytes to the site of zymosan injection in fin was observed in a dose-dependent manner.

SC


Migration of granulocytes was easily observed macroscopically due to transparency of the fin membrane.

zymosan administration.

M AN U


In vivo phagocytic activity of granulocytes that migrated to the fin was observed after

AC C

EP

TE D


Respiratory burst activity of granulocytes at the fin was also detected in vivo.